Blood detecting composition



United States This invention relates to a composition which has utility in the detection of blood. Particularly the invention relates to compositions which are suitable for use in the qualitative detection and quantitative estimation of blood in body fluids such as urine, vomitus, gastric contents, cerebral spinal fluids and in feces.

The detection of occult blood in body fluids and feces has become an invaluable aid to the medical practitioner in the correct diagnosis of a great number of disorders. Blood is found in the gastric contents and in vomitus in conditions associated with erosion of the mucous membranes, in ulcers and in carcinomas. In the feces, the regular and frequent occurrence of occult blood is suggestive of gastro-intestinal cancer, gastric or duodenal ulcers or hemorrhoids. In these conditions, the hemorrhage is often so slight that it is not possible to detect blood by microscopic identifications of the erythrocytes (red blood cells) and a sensitive and specific chemical test for occult blood becomes invaluable. In the urine, blood cells (hematuria) or blood pigment (hemoglobinun'a) is found in typhus, scurvy, purpura, pyernia, nephritis, renal calculi, as the result of a burn covering a large part of the body, by the action of various hemolytic toxins, etc.

The prior art has recognized the need for a simple, reliable test for occult blood. US. Patent No. 2,290,436, issued July 21, 1942, to Kamlet, U.S. Patent No. 2,799,- 660, issued July 16, 1957, to Nicholls and Fonner, and US. Patent No. 2,838,377, issued lune 10, 1958, to Fonner (all assigned to the instant assignee), illustrate various test compositions which have been supplied to meet this need.

The instant inventive concept, like those of the prior art, is based on the catalytic activity of the prosthetic groups present in blood. These catalytically active substances, identified in hemoglobin, belong to the general class of hemoproteins, conjugate proteins all of which have the same prosthetic groups, iron protoporphyrin or haem. This prosthetic group has the ability to catalyze the transfer of oxygen from an oxygen source to an acceptor which in turn becomes oxidized. If the acceptor is a dye precursor, colorless until it becomes oxidized and colored in its oxidized form, then the presence of the catalytic activity is indicated by color formation.

Thus the composition of this invention comprises an indicator or dye precursor plus an oxygen source. In the presence of hemoglobin, which contains the catalytic prosthetic group, the transfer of oxygen from the oxygen source to the acceptor or dye precursor will occur and the indicator will become oxidized and colored. The presence of color, then, is a means of detection of the presence of blood and the rapidity of the color change and the depth or den sity of the color, when compared to a set of standards is a means of the quantitative estimation of the blood present.

In the diagnostic compositions of the prior art the oxygen source has uniformly been of an inorganic nature such as the peroxides, perborates or persul-fates of the alkali or alkaline earth metals. Representative examples include barium peroxide, strontium peroxide, magnesium peroxide, sodium perborosilicate, sodium perborate and stant invention, that an improved composition for detecting the presence of blood may be formed utilizing a suitable dye precursor in combination with organic hydroperoxide.

The systems of the prior art utilizing an inorganic peroxide have been found to require relatively large amounts of peroxide for operability. This is probably because a portion of the inorganic peroxide is consumed by the eatalatic activity of blood. For example, using blood, orthotolidine as an indicator, and an inorganic peroxide as an oxygen source, maximum color development has been found to occur at a. peroxide concentration of 1.5 to 3.0%. With horseradish peroxidase maximum color is obtained at a peroxide concentration of 0.01 to 0.05%. When these relatively large amounts of inorganic peroxides are used the stability of the compositions has been found to be only average since the large amount of inorganic peroxides necessary causes discoloration of the indicator.

It has been found, however, that the organic hydroperoxides of the instant invention act as intact molecules with no decomposition caused by the catalatic activity of the blood. Thus the hydroperoxides are entirely available for the color producing action and only small amounts of the organic hydroperoxides are required. For example, using blood, orthotolidine, and the organic peroxides as an oxygen source, maximum color development is found to exist at peroxide concentrations of 0.05 to 0.10%. This is 30 times more sensitive than the comparable formulation using inorganic peroxides. In addition, the stability of the composition to this invention is improved and the reactivity is greater than identical compositions prepared with the inorganic peroxide of the prior art.

The inventive concept will be more clearly explained by reference to the following illustrative examples.

EXAMPLE 1 Ethyl hydroperoxide was prepared according to the method of Baeyer and Villiger (Baeyer, A., and Villiger, V., Ueber Aethyl Hydroperoxyd, Ber. 34, 73 8-749, 1901). The distillate from the preparation contained ethylhydrogen peroxide in an alcohol-water solution.

Strips of E and D filter paper No. 627, approximately 1 cm. in width and 10 cm. long, were impregnated with a solution containing mg. of barium hydroxide per ml. and were then dried in a vacuum oven. After drying the strips were then overlaid with the distillate of ethyl hydroperoxide (equivalent to 5% hydrogen peroxide) and the sticks were again dried in a vacuum at room temperature. orthotolidine base was dissolved in absolute ethyl alcohol at a concentration of 20 mg. of the indicator per ml. of alcohol and this solution was then placed over the impregnated area. The sticks were again dried in a vacuum oven. Finally a solution of citric acid in ethyl alcohol at a concentration of 151 mg. of citric acid per ml. of alcohol was placed over the impregnated area and the sticks again dried in the vacuum oven.

When dipped in a water dilution of blood containing 1 part of blood per 20,000 parts of water the sticks as pre pared above gave an intense blue color within 30 seconds. In urine, which has an inhibitory efiect on tests for occult blood, a blue color was obtained in a dilution of 1 part of blood to 5,000 parts of urine.

In .an identical composition utilizing an alcohol solution of strontium peroxide as the oxygen source, at the same concentration as above (equivalent to 5% hydrogen peroxide), no color reaction was obtained with a 1/ 20,000

blood in water dilution. It was found necessary to increase the blood concentration to 1:100 before a positive test was obtained. In addition, the composition with the inorganic peroxide was found to be discolored after 1 days storage at room temperature whereas the compositions prepared with the ethyl hydroperoxide were completely operable after a 30 day period of storage.

7 EXAMPLE 2 -1-hydroxycyclohexylhydropenoxide-l was prepared according to the method of Milas, Harris and Panagiotakos (Milas, N. A., I-larnis, S. A. and Panagiotakos, P. C., Studies in Organic Peroxides. VI. Cyclane Peroxides, J. Amer. Chem. Soc. 61, 2430-2432, 1039).

A solution was prepared by admixing together the following components:

100 mg. algin per ml. water 100 mg. gelatin per 5 water 5 ml. of 4 N citrate buffer (pH 4.8) 2 ml. 1% Aquet (wetting agent) Filter paper strips of about 1 cm. by cm. were impregnated with this solution by dipping /2 of the stick into the solution and dried in an oven at 100 C. for two hours.

After drying, a solution of 25 mg. of l-hydroxycycl-ohexylhydroperoxide-l and 20 mg. of o tolidine base per ml. of chloroform was placed over the impregnated area and the sticks redried in the oven.

When the impregnated area of these sticks was immersed in a solution of 1 part of blood to 20,000 parts of water, an intense blue color appeared on the stick within 3-0 seconds. The same blue color was obtained in a urine dilution of blood of 1/ 5,000.

In comparison no color was obtained with an identical composition containing strontium peroxide instead of .the organic peroxide of this example in the same dilution. It was necessary to increase the blood concentration to 1:100 before an equivalent test reaction was ob tained.

EXAMPLE 3 EXAMPLE 4 Following the procedure described in Example 3 above, sticks were prepared utilizing diisopropylbenzene hydroperoxide in the place of cumene hydroperoxide of Example 3.

With dilutions of blood in water as high as 100,000, these sticks gave a blue color within 30 seconds. The same blue color was obtained in urine dilution of 1 part of blood per 20,000 parts of urine.

EXAMPLE 5 Using the procedure described in Example 3 above, sticks were prepared utilizing para-menthane hydroperoxide.

These sticks reacted within 30 seconds to give a blue color with dilutions of blood in water as high as 1:100,- 000. They reacted to give the same blue color in urine dilution of 1 part of blood per 20,000 parts of urine.

EXAMPLE 6 Five gins. of titanium potassium oxalate were dissolved in 1'0 ml. of 30% hydrogen peroxide. The resulting solution was a bright orange. The solution was Washed several times with ether and then driedvto an orange powder in a vacuum oven at a low temperature. The result ing [titanium potassium oxalate hydroperoxide was used to prepare a test composition as follows:

In 10 ml. of water there was dissolved 100 mg. of

gelatin, mg. of algin and 50 mg. of the titanium potassium oxalate hydroperoxide prepared as described above. Filter paper sticks were partially immersed in the resulting solution and were dried in an oven. The impregnated area was then overlaid with a solution of 20 mg. o-tolidine base per ml. of chloroform and were dried in an oven.

The yellow colored sticks resulting gave a green color within 30 seconds when contacted with a dilution of blood in water at a level of 1 part of blood per 10,000 parts of water. In urine dilutions they gave the same green color in a dilution of 1 part of blood per 2,000parts of urine.

The test compositions of Examples 3-6 above were compared with identical compositions in which the organic hydroperoxides of these examples were substituted with strontium peroxide. These comparative sticks containing inorganic peroxides at levels 20 times greater than the amount of organic hydroperoxides used would react with dilutions of blood in urine or water of less than 1 part of blood per 1,000 parts of urine or water. The level of the inorganic peroxides was increased in these comparative sticks until a reaction was obtained at a level of 1 part of blood per 1,000 parts of water. The stability of these test sticks was very poor and within 6 hours they became discolored and unreactive.

The comparative test sticks prepared with inorganic peroxides at a level sufficient to give a reaction at a blood dilution of 1 part per 1,000 parts of water showed a marked efiervescence' when immersed in bacterial catalase solution indicating that the catalatic activity decomposed the inorganic peroxides. There was no color change noted with these sticks. The test compositions of the examples above, however, containing an organic hydroperoxide rapidly turned blue when immersed in the same bacterial catalase solution with no effervescence indicated that the hydroperoxides of this invention were not afiected by the catalatic activity of the solution.

In addition to the orthotolidine indicator or dye precursor which is used in the examples above, it will be appreciated that any indicator material which is capable of accepting oxygen and being oxidized to a colored dye in the presence of the oxygen source and the prosthetic group present in the hemoglobin of blood may be used. For example, such indicators as aniline and its derivatives, o-tolidine, o-toluidine, p-toluidine, o-phenylenediamine, N,N'-dimethyl-p-phenylenediamine, N,N'- diethyl-p-phenylenedianune, benzidine, di-anisidine, ocresol, m-creso'l, p-cresol, alpha naphthol, beta-naphthol, catechol, guaiacol, pyrogallol, etc., may be used.

In addition to the citrate buffer described in connection with the examples above, other bulfers such as tartrate, phosphate, phthalate, acetate and mixtures of these may be used. It is essential that the bufier selected maintain the pH within a range in which the indicator material changes color upon oxidation. Normally, this will be within a pH of about 4 to 7.

In addition to the stabilizers such as gelatin and algin which have been described above, other stabilizing materials such as carogeein, casein, albumin and other of the various protein or polysaccharide materials are operable.

The organic hydroperoxides' which may be used in accordance with the inventive concept have the formula:

R-O'OH wherein R is selected from the group consisting of the following: a

OHz-GH: 0H Own. a. Ca M...

CHr-Cg I The major active ingredients and the operable and Constituent: parts by wt.)

Indicator dye 15-25 Organic hydroperoxide 20-70 Bufier 50-400 Stabilizer -10 To summarize briefly, the instant invention relates to test compositions for the qualitative detection of and the quantitative estimation of occult blood. The compositions comprise an organic hydroperoxide as an oxygen source, a buffer and an indicator material which is capable of accepting the transfer of oxygen from the organic hydroperoxide by the catalytic action of the prosthetic group of hemoglobin of blood. The test compositions are preferably impregnated upon a bibulous carrier such as a filter paper strip, a compression of cellulose fiber, a compres sion of ground cellulose and the like. The test compositions have improved statbility requiring smaller amounts of oxygen sources and are more sensitive than the test compositions of the prior art which utilize inorganic peroxides as the source of oxygen.

What is claimed is:

l. A test composition for the detection of blood which comprises in dry solid form a bibulous carrier upon which is impregnated an organic hydroperoxide selected from the group consisting of ethyl hydroperoxide and l-hydroxycyclohexylhydroperoxide-1 and an indicator material which changes color in the presence of said organic hydroperoxide and blood.

2. A test composition according to claim 1 wherein said organic hydroperoxide is ethyl hydroperoxide.

3. A test composition according to claim 1 wherein said organic hydroperoxide is 1-hydroxycyclohexylhydroperoxide-1.

4. A test composition according to claim 1 wherein said indicator material is an aniline derivative.

5. A test composition according to claim 1 wherein said indicator material is o-tolidine.

6. A test composition according to claim 1 in which the indicator dye is present in a concentration of about from 15 to 25 parts by weight and the organic hydroperoxide in a concentration of about from 20 to 70 parts by Weight.

7. A test composition for the detection of blood which comprises in dry solid form a bibulous carrier upon which is impregnated a composition comprising about from 15 to 25 parts by weight of an indicator dye which is capable of accepting oxygen and being oxidized to a colored dye in the presence of an oxygen source and the prosthetic group of hemoglobin, about from 20 to 70 parts of an organic hydroperoxide selected from the group consisting of ethyl hydroperoxide and l-hydroxycyclohexylhydroperoxide-l, about from 50 O parts of a buffer and about from 5-10 parts of a stabilizer.

References Cited in the fileof this patent UNITED STATES PATENTS 2,223,520 Ioannu Dec. 3, 1940 2,838,377 Fonner June 10, 1958 2,905,594 Morris Sept. 22, 1959 

1. A TEST COMPOSITION FOR THE DETECTION OF BLOOD WHICH COMPRISES IN DRY SOLID FROM A BIBULOUS CARRIER UPON WHICH IS IMPREGNATED AN ORGANIC HYDROPEROXIDE SELECTED FROM THE GROUP CONSISTING OF ETHYL HYDROPEROXIDE AND 1=HYDROXYCYCLOHEXYLHYDROPEROXIDE-1 AND AN INDICATOR MATERIAL WHICH CHANGES COLOR IN THE PRESENCE OF SAID ORGANIC HYDDROPEROXIDE AND BLOOD. 